Abstract 224: The Tyrosine Kinase Arg Mediates Endothelial Barrier Disruption by Modulating Integrin Distribution

Abstract

Introduction Endothelial barrier disruption and vascular leakage during inflammation forms an important threat to the critically ill. In a recent study we demonstrated that the tyrosine kinase inhibitor imatinib effectively attenuates endothelial barrier disruption via inhibition of the kinase Abl-related gene (ARG). Here, we evaluate how ARG mediates endothelial barrier disruption. Methods & Results ARG was depleted from endothelial cells with various siRNAs, yielding >85% reduction in ARG protein expression. Disruption of the endothelial barrier by the inflammatory mediator thrombin was lower in ARG-depleted cells than in control cells (30% versus 60% drop in endothelial resistance). ARG deficient mice showed a significant reduction of VEGF-induced vascular leakage, stressing the in vivo contribution of ARG to vascular leakage. ARG was activated upon endothelial activation in vitro , as indicated by a 2-3 fold increase in CrkL phosphorylation, the downstream target of ARG. Surprisingly, ARG depletion resulted in 40% reduction in VE-cadherin protein expression. At the same time, ARG-depleted cells completely lacked a contractile response to inflammatory mediators, despite normal myosin light chain phosphorylation. ARG depletion resulted in increased protein expression of α 5 β 1 integrin, increased expression of β 1 integrin at the cell surface and redistribution of α 5 β 1 integrin to the cell edges (close to cell-cell contacts). These signaling events were associated with increased adhesive force of ARG-depleted endothelial cells to the extracellular matrix. The clinical relevance of ARG was confirmed by increased amounts of phosphorylated CrkL in the pulmonary endothelium of septic patients. Conclusion This study shows that ARG is a significant mediator of endothelial barrier disruption during inflammation. ARG regulates integrin dynamics during endothelial barrier disruption, and the ARG-induced changes in integrin dynamics contribute to endothelial barrier disruption.